Detection and treatment of cancers of the colon

ABSTRACT

The present invention relates to compositions and methods for cancer therapies and diagnostics, including but not limited to, cancer markers. In particular, the present invention provides tumor antigens associated with specific cancers and diagnostic assays for the detection of such antigens and associated autoantibodies as indicative of the presence of specific cancers. The present invention further provides cancer immunotherapy utilizing the tumor antigens of the present invention.

This application claims priority to provisional patent application Ser.No. 60/469,717, filed May 12, 2003, which is herein incorporated byreference in its entirety.

This invention was made in part with government support under Grant No.CA084953 awarded by the National Cancer Institute. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for cancertherapies and diagnostics, including but not limited to, cancer markers.In particular, the present invention provides tumor antigens associatedwith specific cancers and diagnostic assays for the detection of suchantigens and associated autoantibodies as indicative of the presence ofspecific cancers.

BACKGROUND OF THE INVENTION

The term cancer collectively refers to more than 100 different diseasesthat affect nearly every part of the body. Throughout life, healthycells in the body divide, grow, and replace themselves in a controlledfashion. Cancer starts when the genes directing this cellular divisionmalfunction, and cells begin to multiply and grow out of control. A massor clump of these abnormal cells is called a tumor. Not all tumors arecancerous. Benign tumors, such as moles, stop growing and do not spreadto other parts of the body. But cancerous, or malignant, tumors continueto grow, crowding out healthy cells, interfering with body functions,and drawing nutrients away from body tissues. Malignant tumors canspread to other parts of the body through a process called metastasis.Cells from the original tumor break off, travel through the blood orlymphatic vessels or within the chest, abdomen or pelvis, depending onthe tumor, and eventually form new tumors elsewhere in the body.

Only 5-10% of cancers are thought to be hereditary. The rest of thetime, the genetic mutation that leads to the disease is brought on byother factors. The most common cancers are linked to smoking, sunexposure, and diet. These factors, combined with age, family history,and overall health, contribute to an individual's cancer risk.

Several diagnostic tests are used to rule out or confirm cancer. Formany cancers, the most definitive way to do this is to take a smallsample of the suspect tissue and look at it under a microscope—thisprocess is called a biopsy. However, many biopsies are invasive,unpleasant procedures with their own associated risks, such as pain,bleeding, infection, and tissue or organ damage. In addition, if abiopsy does not result in an accurate or large enough sample, a falsenegative or misdiagnosis can result, often required that the biopsy berepeated. What is needed in the art are improved methods to specificallydetect, characterize, and monitor specific types of cancer.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for cancertherapies and diagnostics, including but not limited to, cancer markers.In particular, the present invention provides tumor antigens associatedwith specific cancers and diagnostic assays for the detection of suchantigens and associated autoantibodies as indicative of the presence ofspecific cancers.

There is increasing evidence for an immune response to cancer in humans,demonstrated by the identification of autoantibodies to tumor antigens(Stocket et al., J. Exp. Med., 187:1349 [1998]; Boon and Old, Curr.Opin. Immunol. 9:681 [1997]; Soussl, Cancer Res. 60:1777 [2000]; Old andChen, J. Exp. Med. 187:1163 [1998]). The identification of panels oftumor antigens that elicit a humoral response has utility in cancerscreening, diagnosis, and in establishing a prognosis. Such antigensalso have utility in immunotherapy against cancers. Several approachesare currently available for the identification of tumor antigens. Thepresent invention provides a proteomic-based approach for theidentification of tumor antigens that induce an antibody response. Incontrast to other approaches based on the analysis of recombinantproteins, a proteomic approach allows identification of autoantibodiesto proteins that are directly derived from cancer cells or tumors andthus may uncover antigenicity associated with post-translationalmodification.

To date, discovery of autoantibodies has been limited by the detectiontechnology. In some embodiments, the present invention provides acombination of liquid-phase protein separation and protein microarraytechnology that provides an effective means to array a wide repertoireof tumor cell proteins derived from the tumor type of interest. As aresult, detection of specific interactions between tumor cell antigensin individual fractions and antibodies in patient sera is substantiallyfacilitated. Experiments conducted during the course of development ofthe present invention comprising hybridization of these proteinmicroarrays with sera from cancer patients and controls resulted in thedetection of a fraction (L04428) that exhibited a high frequency ofimmunoreactivity with colon cancer sera. This fraction was analyzed byQ-TOF tandem mass spectrometry, and was found to contain UCH-L3.Subsequent independent analysis by means of 2-D PAGE and Westernblotting uncovered autoantibodies against UCH-L3 in sera from 19/43newly diagnosed patients with colon cancer. However, no antibodies weredetected in sera obtained from 15 healthy individuals, 15 patients withcolon adenomas, and 24 patients with lung cancer.

Another member of the UCHL family, UCH-L1, was previously identified asan antigen that induces an antibody response in lung cancer (Brichory etal., Cancer Research 61, 7908-7912 [2001]). UCH-L1 is widely expressedin neuronal tissues at all stages of neuronal differentiation, and maybe expressed during neuroendocrine differentiation of lung cancer.Ubiquitination and targeting of cellular proteins for subsequentdegradation via ubiquitin-mediated proteolysis is an important mechanismregulating a broad spectrum of cellular processes. In tumors, increaseddeubiquitination of cyclins by UCH-L1 may contribute to the uncontrolledgrowth of somatic cells (Hibi et al., American Journal of Pathology 155,711-715 [1999]; Kurihara et al., Human Molecular Genetics 10, 1963-1970[2001]; Tezel et al., Clinical Cancer Research 6, 4764-4767 [2000]).

The methods of the present invention allow protein microarray screeningof patient sera to determine reactivity with individual proteinfractions. A humoral response directed against UCH-L3, detectable inboth the LoVo colon adenocarcinoma cell line and in colon tumors,occurred in 44% of newly diagnosed patients with colon adenocarcinoma.DNA microarray analysis revealed that UCH-L3 was expressed atapproximately 3-5 fold higher levels in colon tumors than that observedin all other tumor types examined. These findings are in contrast tonormal expression of UCH-L3, whose mRNA is highly enriched in heart,skeletal muscle and testis (Wada et al., Biochem. Biophys. Res. Commun.251,688-692 [1998]), but much lower in all other tissues, thusdemonstrating aberrant expression of UCH-L3 in colon cancer.Accordingly, in some embodiments, the present invention provides methodsof detecting autoantibodies to the UCH-L3 as a biomarker for coloncancer.

In other embodiments, UCH-L3 expression is analyzed. In yet otherembodiments, the existence of one or more other markers is determined tocharacterize the presence, absence, or stage of colon cancer in asubject. For example, in some embodiments, the present inventionprovides a method for detecting cancer, comprising providing a samplefrom a subject suspected of having cancer; and detecting the presence ofUCH-L3 in the sample, thereby detecting cancer. In some embodiments, thecancer is colorectal cancer. In some embodiments, the subject comprisesa human subject. In some embodiments, the sample comprises a bloodsample or a tumor sample. In some embodiments, detecting comprisesexposing the sample to an antibody and detecting the antibody binding toUCH-L3. In other embodiments, detecting comprises detecting the presenceof an autoantibody to UCH-L3 (e.g., exposing the sample to anautoantibody specific antibody and detecting the autoantibody specificantibody binding to the antibody). In some embodiments, the methodfurther comprises step c) providing a prognosis to the subject. In someembodiments, detecting cancer further comprises detecting a stage of thecancer. In some embodiments, detecting cancer further comprisesdetecting a sub-type of the cancer.

In other embodiments, the present invention provides a kit for detectingthe presence of cancer in a subject, comprising a reagent capable ofspecifically detecting the presence of UCH-L3; and instructions forusing the kit for detecting the presence of cancer in the subject. Insome embodiments, the antibody is a UCH-L3 specific antibody. In otherembodiments, the antibody is an antibody specific for an autoantibody toUSCH-L3.

In yet other embodiments, the present invention provides a method foreliciting a cancer specific immune response, comprising providing animmunogenic composition comprising UCH-L3 tumor antigen; and a subjectdiagnosed with a cancer; and administering the immunogenic compositionto the subject under conditions such that the subject generates animmune response to the cancer. In some embodiments, the immunogeniccomposition further comprises an immune enhancing cytokine. In someembodiments, the immune enhancing cytokine is expressed by a cell. Insome embodiments, the immune response results in a detectable decreasein the presence of the cancer. In other embodiments, the immune responseresults in a measurable decrease in the level of the UCH-L3 tumorantigen. In still other embodiments, the immune response results in ameasurable decrease in the level of autoantibodies to the UCH-L3 tumorantigen. In some embodiments, the cancer is colorectal cancer. In someembodiments, the subject is a human.

In still further embodiments, the present invention provides a method oftreating cancer in a subject, comprising providing a subject; and atherapeutic composition comprising an antibody directed toward UCH-L3;and administering the therapeutic composition to the subject. In someembodiments, the cancer is colorectal cancer. In some embodiments, theantibody is attached to a cytotoxic agent (e.g., including, but notlimited to, of chemotherapeutic agents, radioisotopes, cytosines,cytokines, and toxins). In certain embodiments, the cytotoxic agent isRicin A chain.

In some preferred embodiments the caner markers of the present inventionare detected in combination with other colon cancer markers to provide amore informative profile (See e.g., U.S. Pat. No. 6,448,041 and U.S.Patent Publication Nos. 20030073105, 20030008284, and 20020160382; eachof which is herein incorporated by reference in its entirety).

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular antibody.

When a protein or fragment of a protein is used to immunize a hostanimal, numerous regions of the protein may induce the production ofantibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as “antigenic determinants”. An antigenic determinantmay compete with the intact antigen (i.e., the “immunogen” used toelicit the immune response) for binding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “tumor antigen” refers to an immunogenicepitope (e.g., protein) expressed by a tumor cell. The protein may beexpressed by non tumor cells but be immunogenic only when expressed by atumor cell. Alternatively, the protein may be expressed by tumor cells,but not normal cells.

As used herein, the term “autoantibody” refers to an antibody producedby a host (with or without immunization) and directed to a host antigen(e.g., a tumor antigen).

As used herein, the term “cancer vaccine” refers to a composition (e.g.,a tumor antigen and a cytokine) that elicits a tumor-specific immuneresponse. The response is elicited from the subject's own immune systemby administering the cancer vaccine composition at a site (e.g., a sitedistant from the tumor). In preferred embodiments, the immune responseresults in the eradication of tumor cells everywhere in the body (e.g.,both primary and metastatic tumor cells).

As used herein, the term “host” refers to any animal (e.g., a mammal),including, but not limited to, humans, non-human primates, rodents, andthe like, that is to be the recipient of a particular treatment.Typically, the terms “host” and “patient” are used interchangeablyherein in reference to a human subject.

As used herein, the term “immune-enhancing cytokine” refers to acytokine that is capable of enhancing the immune response when thecytokine is generated in situ or is administered to a mammalian host.Immune enhancing cytokines include, but are not limited to,granulocyte-macrophage colony stimulating factor, interleukin-2,interleukin-3, interleukin-4, and interleukin-12.

As used herein, the term “subject suspected of having cancer” refers toa subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass). A subject suspected of having cancermay also have on or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has receivedan initial diagnosis (e.g., a CT scan showing a mass) but for whom thesub-type or stage of cancer is not known. The term further includespeople who once had cancer (e.g., an individual in remission).

As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, genetic predisposition,environmental expose, preexisting non-cancer diseases, and lifestyle.

As used herein, the term “stage of cancer” refers to a numericalmeasurement of the level of advancement of a cancer. Criteria used todetermine the stage of a cancer include, but are not limited to, thesize of the tumor, whether the tumor has spread to other parts of thebody and where the cancer has spread (e.g., within the same organ orregion of the body or to another organ).

As used herein, the term “sub-type of cancer” refers to different typesof cancer that effect the same organ (ductal cancer, lobular cancer, andinflammatory breast cancer are sub-types of breast cancer.

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of cancer (e.g., asdetermined by the diagnostic methods of the present invention) on asubject's future health (e.g., expected morbidity or mortality).

As used herein, the term “detecting the presence of cancer in a subject”refers to detecting the presence of a tumor antigen or autoantibodyindicative of cancer. In preferred embodiments, the detecting involvesthe diagnostic methods of the present invention.

As used herein, the term “cancer-specific immune response” refers to animmune response directed to a cancerous cell, or, in particular, a tumorantigen expressed by the cancerous cell.

As used herein, the term “subject diagnosed with a cancer” refers to asubject having cancerous cells. The cancer may be diagnosed using anysuitable method, including but not limited to, the diagnostic methods ofthe present invention.

As used herein, the term “detectable decrease in the presence of saidcancer” refers to a measurable decrease in diagnostic symptoms of acancer (e.g., size of a tumor or lack of tumor antigen expression).

As used herein, the term “non-human animals” refers to all non-humananimals. Such non-human animals include, but are not limited to,vertebrates such as rodents, non-human primates, ovines, bovines,ruminants, lagomorphs, porcines, caprines, equines, canines, felines,aves, etc.

As used herein, the term “gene targeting” refers to the alteration ofgenes through molecular biology techniques. Such gene targetingincludes, but is not limited to, generation of mutant genes and knockoutgenes through recombination. When a gene is altered such that itsproduct is no longer biologically active in a wild-type fashion, themutation is referred to as a “loss-of-function” mutation. When a gene isaltered such that a portion or the entirety of the gene is deleted orreplaced, the mutation is referred to as a “knockout” mutation.

As used herein, the term “gene transfer system” refers to any means ofdelivering a composition comprising a nucleic acid sequence to a cell ortissue. For example, gene transfer systems include, but are not limitedto, vectors (e.g., retroviral, adenoviral, adeno-associated viral, andother nucleic acid-based delivery systems), microinjection of nakednucleic acid, polymer-based delivery systems (e.g., liposome-based andmetallic particle-based systems), biolistic injection, and the like. Asused herein, the term “viral gene transfer system” refers to genetransfer systems comprising viral elements (e.g., intact viruses andmodified viruses) to facilitate delivery of the sample to a desired cellor tissue. As used herein, the term “adenovirus gene transfer system”refers to gene transfer systems comprising intact or altered virusesbelonging to the family Adenoviridae.

As used herein, the term “site-specific recombination target sequences”refers to nucleic acid sequences that provide recognition sequences forrecombination factors and the location where recombination takes place.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule including, but not limited to DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (e.g., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (e.g., RNA or protein), while “down-regulation” or “repression”refers to regulation that decreases production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides or polynucleotidesin a manner such that the 5′ phosphate of one mononucleotide pentosering is attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotide orpolynucleotide is referred to as the “5′ end” if its 5′ phosphate is notlinked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequentmononucleotide pentose ring. As used herein, a nucleic acid sequence,even if internal to a larger oligonucleotide or polynucleotide, also maybe said to have 5′ and 3′ ends. In either a linear or circular DNAmolecule, discrete elements are referred to as being “upstream” or 5′ ofthe “downstream” or 3′ elements. This terminology reflects the fact thattranscription proceeds in a 5′ to 3′ fashion along the DNA strand. Thepromoter and enhancer elements that direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element or the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequence thatencodes a gene product. The coding region may be present in a cDNA,genomic DNA or RNA form. When present in a DNA form, the oligonucleotideor polynucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 200 residues long (e.g., between 15 and 100), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defined infra).

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (T. Maniatis et al., Science 236:1237 [1987]). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells, andviruses (analogous control elements, i.e., promoters, are also found inprokaryote). The selection of a particular promoter and enhancer dependson what cell type is to be used to express the protein of interest. Someeukaryotic promoters and enhancers have a broad host range while othersare functional in a limited subset of cell types (for review, See e.g.,Voss et al., Trends Biochem. Sci., 11:287 [1986]; and T. Maniatis etal., supra). For example, the SV40 early gene enhancer is very active ina wide variety of cell types from many mammalian species and has beenwidely used for the expression of proteins in mammalian cells (Dijkemaet al., EMBO J. 4:761 [1985]). Two other examples of promoter/enhancerelements active in a broad range of mammalian cell types are those fromthe human elongation factor 1α gene (Uetsuki et al., J. Biol. Chem.,264:5791 [1989]; Kim et al., Gene 91:217 [1990]; and Mizushima andNagata, Nuc. Acids. Res., 18:5322 [1990]) and the long terminal repeatsof the Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA79:6777 [1982]) and the human cytomegalovirus (Boshart et al., Cell41:521 [1985]). Some promoter elements serve to direct gene expressionin a tissue-specific manner.

As used herein, the term “promoter/enhancer” denotes a segment of DNAwhich contains sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element, see above for a discussion of these functions). Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An “endogenous” enhancer/promoter is onethat is naturally linked with a given gene in the genome. An “exogenous”or “heterologous” enhancer/promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques such as cloning and recombination) suchthat transcription of that gene is directed by the linkedenhancer/promoter.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (J. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a DNA sequence that directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one that is isolated from onegene and placed 3′ of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation (J. Sambrook, supra, at 16.6-16.7).

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequencesthat allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors thatcontain either the SV40 or polyoma virus origin of replication replicateto high “copy number” (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen. Vectors that contain the replicons frombovine papillomavirus or Epstein-Barr virus replicate extrachromosomallyat “low copy number” (˜100 copies/cell).

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence“5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is a nucleic acid molecule that at leastpartially inhibits a completely complementary nucleic acid molecule fromhybridizing to a target nucleic acid is “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous nucleic acid molecule to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target that issubstantially non-complementary (e.g., less than about 30% identity); inthe absence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m)value may be calculated by theequation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With “high stringency” conditions, nucleicacid base pairing will occur only between nucleic acid fragments thathave a high frequency of complementary base sequences. Thus, conditionsof “weak” or “low” stringency are often required with nucleic acids thatare derived from organisms that are genetically diverse, as thefrequency of complementary sequences is usually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄·H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄·H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄·H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.)(see definition above for “stringency”).

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]).This amplification enzyme will not replicate other nucleic acid.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters (Chamberlin et al.,Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme willnot ligate the two oligonucleotides or polynucleotides, where there is amismatch between the oligonucleotide or polynucleotide substrate and thetemplate at the ligation junction (Wu and Wallace, Genomics 4:560[1989]). Finally, Taq and Pfu polymerases, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor primerhybridization with the target sequences and not hybridization withnon-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press[1989]).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”.In contrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” refers to the region of nucleic acidbounded by the primers. Thus, the “target” is sought to be sorted outfrom other nucleic acid sequences. A “segment” is defined as a region ofnucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195 4,683,202, and4,965,188, hereby incorporated by reference, which describe a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process are, themselves, efficient templatesfor subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The terms “in operable combination,” “in operable order,” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

“Amino acid sequence” and terms such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is,the native protein contains only those amino acids found in the proteinas it occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58 [1989]).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52[1989]).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

The term “transgene” as used herein refers to a foreign gene that isplaced into an organism by, for example, introducing the foreign geneinto newly fertilized eggs or early embryos. The term “foreign gene”refers to any nucleic acid (e.g., gene sequence) that is introduced intothe genome of an animal by experimental manipulations and may includegene sequences found in that animal so long as the introduced gene doesnot reside in the same location as does the naturally occurring gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” Vectorsare often derived from plasmids, bacteriophages, or plant or animalviruses.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome-binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher (or greater) than thatobserved in a given tissue in a control or non-transgenic animal. Levelsof mRNA are measured using any of a number of techniques known to thoseskilled in the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the mRNA-specificsignal observed on Northern blots). The amount of mRNA present in theband corresponding in size to the correctly spliced transgene RNA isquantified; other minor species of RNA which hybridize to the transgeneprobe are not considered in the quantification of the expression of thetransgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973]),has been modified by several groups to optimize conditions forparticular types of cells. The art is well aware of these numerousmodifications.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

As used herein, the term “selectable marker” refers to the use of a genethat encodes an enzymatic activity that confers the ability to grow inmedium lacking what would otherwise be an essential nutrient (e.g. theHIS3 gene in yeast cells); in addition, a selectable marker may conferresistance to an antibiotic or drug upon the cell in which theselectable marker is expressed. Selectable markers may be “dominant”; adominant selectable marker encodes an enzymatic activity that can bedetected in any eukaryotic cell line. Examples of dominant selectablemarkers include the bacterial aminoglycoside 3′ phosphotransferase gene(also referred to as the neo gene) that confers resistance to the drugG418 in mammalian cells, the bacterial hygromycin G phosphotransferase(hyg) gene that confers resistance to the antibiotic hygromycin and thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpt gene) that confers the ability to grow in thepresence of mycophenolic acid. Other selectable markers are not dominantin that there use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominant selectable markersinclude the thymidine kinase (tk) gene that is used in conjunction withtk⁻ cell lines, the CAD gene, which is used in conjunction withCAD-deficient cells, and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene, which is used in conjunctionwith hprt-cell lines. A review of the use of selectable markers inmammalian cell lines is provided in Sambrook, J. et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, New York (1989) pp. 16.9-16.15.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, transformed celllines, finite cell lines (e.g., non-transformed cells), and any othercell population maintained in vitro.

As used, the term “eukaryote” refers to organisms distinguishable from“prokaryotes.” It is intended that the term encompass all organisms withcells that exhibit the usual characteristics of eukaryotes, such as thepresence of a true nucleus bounded by a nuclear membrane, within whichlie the chromosomes, the presence of membrane-bound organelles, andother characteristics commonly observed in eukaryotic organisms. Thus,the term includes, but is not limited to such organisms as fungi,protozoa, and animals (e.g., humans).

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that is a candidate for use to treat or prevent adisease, illness, sickness, or disorder of bodily function. Testcompounds comprise both known and potential therapeutic compounds. Atest compound can be determined to be therapeutic by screening using thescreening methods of the present invention.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for cancertherapies and diagnostics, including but not limited to, cancer markers.In particular, the present invention provides tumor antigens associatedwith specific cancers and diagnostic assays for the detection of suchantigens and associated autoantibodies as indicative of the presence ofspecific cancers (e.g., colorectal cancer).

In the United States, colorectal cancer is the second leading cause ofall cancer deaths. In most cases colorectal cancer strikes men and womenover age 50. If the cancer is found and treated early before it spreadsto lymph nodes or other organs, the survival rate is higher. However,less than 40% of colon cancers are discovered at an early stage a timewhere interventions have a greater chance of success and where moreoptions are available.

Age and health history can affect the risk of developing colon cancer.Risk factors include an age of 50 or older, a family history of cancerof the colon or rectum, a personal history of cancer of the colon,rectum, ovary, endometrium, or breast, a history of polyps (smallnoncancerous growths) in the colon, a history of ulcerative colitis(ulcers in the lining of the large intestine), and certain hereditaryconditions, such as familial adenomatous polyposis and hereditarynonpolyposis colon cancer (HNPCC; Lynch Syndrome).

Colon cancer is diagnosed by fecal occult blood test, digital rectalexamination, barium enema, or sigmoidocopy or colonoscopy. Treatmentoptions and prognosis depend on the stage of the cancer (whether thecancer is in the inner lining of the colon only, involves the wholecolon, or has spread to other places in the body) and the patient'sgeneral health. Treatment options include surgery (sometimes includingcolostomy), chemotherapy, and radiation.

The currently available diagnostic techniques are limited in theirability to decisively identify and characterize tumors. In view of thelimitations of current cancer detection technologies, what are neededare tumor-specific markers that can be used to detect early stagecolorectal cancers (e.g., tumors too small to be detected byconventional techniques) and can provide information about themorphology of the cancer. In addition, the art is in need of effectivetreatments for colorectal cancer.

The present invention thus provides improved diagnostic and treatmentmethods directed toward a specific cancer. The description below isdivided into the following sections: I) identification of tumorantigens, II) antibodies, III) detection of tumor antigens, IV) cancerimmunotherapy, V) other therapies and VI) transgenic animals.

I. Identification of Tumor Antigens

In some embodiments, the present invention provides a gelelectrophoresis technique useful in the separation, identification, andcharacterization of tumor antigens. The technique is configured toidentify antigens associated with a specific tumor type. Experimentsconducted during the development of the present invention identified aseries of tumor antigens specifically associated with cancer.

A. Separation and Identification Techniques

In some embodiments, proteins from non-cancerous and cancerous cells(and/or tissues) are separated using an established two-dimensional(2-D) PAGE procedure (See e.g., Strahler et al., 1989. ProteinStructure: A practical approach, T. E. Creighton ed., IRL Press,England, pgs. 65-92). Briefly, cells and tissues are solubilized inlysis buffer containing carrier ampholytes. Proteins are then applied toisoelectric focusing gels and separated based on isoelectric point. Thefirst-dimension gel is then loaded onto the second dimension gel(acrylamide gradient). Proteins are then transferred to a PVDF membranefor Western blotting or visualized by silver-staining of the acrylamidegradient gels. In some embodiments, proteins separated by 2-D PAGE arecharacterized using Western blotting. Following transfer to PVDFmembranes, the membranes are incubated with serum obtained from patientsor from controls and bound antibodies are visualized.

In some embodiments, proteins separated by 2-D PAGE are silver stainedto visualize proteins. The proteins of interest are excised from the 2-Dgels, purified, and digested with trypsin. Digested proteins are thenanalyzed using matrix assisted laser desorption ionization-time offlight (MALDI-TOF) mass spectroscopy. In preferred embodiments, proteinsof particular interest are identified. In some embodiments, proteins areidentified by using the search program MS-Fit (University of California,available at prospector.ucsf.edu) to search for proteins in the databaseNCBI.

In other embodiments, following 2-D separation, proteins are placed onprotein microarrays. The microarrays are then probed with patient serumto identify autoantibodies. Protein microarrays may be generated usingany suitable method including, but not limited to, those disclosedherein (See e.g., Experimental Section).

B. Identification of Autoantibodies

The 2-D analysis described above was used to identify proteins thatelicited humoral immune responses in colorectal cancer patients but notnormal patients (See Experimental section). In particular, UCH-L3 wasidentified. The detection of UCH-L3 finds utility in the diagnosis andcharacterization of colorectal cancer, as described below.

II. Antibodies

The present invention provides isolated antibodies. In preferredembodiments, the present invention provides monoclonal antibodies thatspecifically bind to an isolated polypeptide comprised of at least fiveamino acid residues of tumor antigens. In other embodiments, the presentinvention provides antibodies that recognize autoantibodies to the tumorantigens. These antibodies find use in the diagnostic and therapeuticmethods described below.

An antibody against a protein of the present invention may be anymonoclonal or polyclonal antibody, as long as it can recognize theprotein. Antibodies can be produced by using a protein of the presentinvention as the antigen according to a conventional antibody orantiserum preparation process.

The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animalwhose antibody titer has been confirmed (e.g., a mouse) is selected, and2 days to 5 days after the final immunization, its spleen or lymph nodeis harvested and antibody-producing cells contained therein are fusedwith myeloma cells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can be carriedout, for example, by reacting the labeled protein, as describedhereinafter and antiserum and then measuring the activity of thelabeling agent bound to the antibody. The cell fusion can be carried outaccording to known methods, for example, the method described by Koehlerand Milstein (Nature 256:495 [1975]). As a fusion promoter, for example,polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and the like.The proportion of the number of antibody producer cells (spleen cells)and the number of myeloma cells to be used is preferably about 1:1 toabout 20:1. PEG (preferably PEG 1000-PEG 6000) is preferably added inconcentration of about 10% to about 80%. Cell fusion can be carried outefficiently by incubating a mixture of both cells at about 20° C. toabout 40° C., preferably about 30° C. to about 37° C. for about 1 minuteto 10 minutes.

Various methods may be used for screening for a hybridoma producing theantibody (e.g., against a tumor antigen or autoantibody of the presentinvention). For example, where a supernatant of the hybridoma is addedto a solid phase (e.g., microplate) to which antibody is adsorbeddirectly or together with a carrier and then an anti-immunoglobulinantibody (if mouse cells are used in cell fusion, anti-mouseimmunoglobulin antibody is used) or Protein A labeled with a radioactivesubstance or an enzyme is added to detect the monoclonal antibodyagainst the protein bound to the solid phase. Alternately, a supernatantof the hybridoma is added to a solid phase to which ananti-immunoglobulin antibody or Protein A is adsorbed and then theprotein labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase.

Selection of the monoclonal antibody can be carried out according to anyknown method or its modification. Normally, a medium for animal cells towhich HAT (hypoxanthine, aminopterin, thymidine) are added is employed.Any selection and growth medium can be employed as long as the hybridomacan grow. For example, RPMI 1640 medium containing 1% to 20%, preferably10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetalbovine serum, a serum free medium for cultivation of a hybridoma(SFM-101, Nissui Seiyaku) and the like can be used. Normally, thecultivation is carried out at 20° C. to 40° C., preferably 37° C. forabout 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO₂gas. The antibody titer of the supernatant of a hybridoma culture can bemeasured according to the same manner as described above with respect tothe antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody (e.g., against atumor antigen or autoantibody of the present invention) can be carriedout according to the same manner as those of conventional polyclonalantibodies such as separation and purification of immunoglobulins, forexample, salting-out, alcoholic precipitation, isoelectric pointprecipitation, electrophoresis, adsorption and desorption with ionexchangers (e.g., DEAE), ultracentrifugation, gel filtration, or aspecific purification method wherein only an antibody is collected withan active adsorbent such as an antigen-binding solid phase, Protein A orProtein G and dissociating the binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared and an animal is immunized bythe complex according to the same manner as that described with respectto the above monoclonal antibody preparation. A material containing theantibody is recovered from the immunized animal and the antibody isseparated and purified.

As to the complex of the immunogen and the carrier protein to be usedfor immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to an hapten in a weight ratio of about 0.1 part to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten.

In addition, various condensing agents can be used for coupling of ahapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide-activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like,of an animal immunized by the above method. The antibody titer in theantiserum can be measured according to the same manner as that describedabove with respect to the supernatant of the hybridoma culture.Separation and purification of the antibody can be carried out accordingto the same separation and purification method of immunoglobulin as thatdescribed with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, a tumor antigen of thepresent invention (further including a gene having a nucleotide sequencepartly altered) can be used as the immunogen. Further, fragments of theprotein may be used. Fragments may be obtained by any methods including,but not limited to expressing a fragment of the gene, enzymaticprocessing of the protein, chemical synthesis, and the like.

III. Detection of Tumor Antigens

As described above, the presence of an immune response to specificproteins expressed in cancerous cells is indicative of the presence ofcancer. Accordingly, in some embodiments, the present invention providesmethods (e.g., diagnostic methods) for detecting the presence of tumorantigens. In some embodiments (e.g., where tumor antigens are expressedin cancerous cells but not non-cancerous cells), tumor antigen proteinsare detected directly. In other embodiments (e.g., where the presence ofan autoantibody in cancerous but not cancerous cells is indicative ofthe presence of cancer), autoantibodies to the tumor antigens aredetected. In preferred embodiments, tumor antigens are detected directlyin tumors or cells suspected of being cancerous.

The diagnostic methods of the present invention find utility in thediagnosis and characterization of cancers. For example, the presence ofan autoantibody to a specific protein may be indicative of a cancer. Inaddition, certain autoantibodies may be indicative of a specific stageor sub-type of the same cancer.

The information obtained is used to determine prognosis and appropriatecourse of treatment. For example, it is contemplated that individualswith a specific autoantibody or stage of cancer may respond differentlyto a given treatment than individuals lacking the antibody. Theinformation obtained from the diagnostic methods of the presentinvention thus provides for the personalization of diagnosis andtreatment.

A. Detection of Antigens

In some embodiments, antibodies are used to detect tumor antigens in abiological sample from an individual. The biological sample can be abiological fluid, such as, but not limited to, blood, serum, plasma,interstitial fluid, urine, cerebrospinal fluid, and the like, containingcells. In preferred embodiments, the biological sample comprises cellssuspected of being cancerous (e.g., cells obtained from a biopsy).

The biological samples can then be tested directly for the presence oftumor antigens using an appropriate strategy (e.g., ELISA orradioimmunoassay) and format (e.g., microwells, dipstick (e.g., asdescribed in International Patent Publication WO 93/03367), etc).Alternatively, proteins in the sample can be size separated (e.g., bypolyacrylamide gel electrophoresis (PAGE), in the presence or not ofsodium dodecyl sulfate (SDS), and the presence of tumor antigensdetected by immunoblotting (e.g., Western blotting). Immunoblottingtechniques are generally more effective with antibodies generatedagainst a peptide corresponding to an epitope of a protein, and hence,are particularly suited to the present invention.

Antibody binding is detected by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay.)

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays are well known in the art (See e.g.,U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each ofwhich is herein incorporated by reference). In some embodiments, theanalysis and presentation of results is also automated. For example, insome embodiments, software that generates a prognosis based on thepresence or absence of a series of antigens is utilized.

B. Detection of Autoantibodies

In some embodiments, the presence of autoantibodies to a tumor antigenis detected. This approach to diagnosing and typing tumors isparticularly suited to tumor antigens that are present, but notimmunogenic, in normal cells and immunogenic in tumor cells. Forexample, in some embodiments, antibodies (e.g., monoclonal orpolyclonal) are generated to the autoantibodies identified during thedevelopment of the present invention. Such antibodies are then used todetect the presence of autoantibodies using any suitable technique,including but not limited to, those described above.

In other embodiments, tumor proteins are attached to a solid surface.The presence of autoantibodies is identified by contacting the solidsurface (e.g., microarray) with serum from the subject and detectingbinding to a tumor marker. One exemplary method for performing such anassay is described in the experimental section below.

C. Detection Kits

The present invention further provides kits for the diagnosis and typingof cancer. In some embodiments, the kits contain antibodies specific fora tumor antigen or autoantibody, in addition to detection reagents andbuffers. In preferred embodiments, the kits contain all of thecomponents necessary to perform a detection assay, including allcontrols, directions for performing assays, and any necessary softwarefor analysis and presentation of results.

D. Other Detection Methods

The present invention is not limited to the detection methods describedabove. Any suitable detection method that allows for the specificdetection of cancerous cells may be utilized. For example, in someembodiments, the expression of RNA corresponding to a tumor antigen geneis detected by hybridization to an antisense oligonucleotide (e.g.,those described below). In other embodiments, RNA expression is detectedby hybridization assays such as Northern blots, RNase assays, reversetranscriptase PCR amplification, and the like.

In further embodiments of the present invention, the presence ofparticular sequences in the genome of a subject are detected. Suchsequences include tumor antigen sequences associated with abnormalexpression of tumor antigens (e.g., overexpression or expression at aphysiological inappropriate time). These sequences includepolymorphisms, including polymorphisms in the transcribed sequence(e.g., that effect tumor antigen processing and/or translation) andregulatory sequences such as promoters, enhances, repressors, and thelike. These sequences may also include polymorphisms in genes or controlsequences associated with factors that affect expression such astranscription factors, and the like. Any suitable method for detectingand/or identifying these sequences is within the scope of the presentinvention including, but not limited to, nucleic acid sequencing,hybridization assays (e.g., Southern blotting), single nucleotidepolymorphism assays (See e.g., U.S. Pat. No. 5,994,069, hereinincorporated by reference in its entirety), and the like.

Direct and/or indirect measures of tumor antigen expression may be usedas a marker within the scope of the present invention. Because thepresent invention provides a link between tumor antigen expression andcancer, any indication of tumor expression may be used. For example, theexpression, activation, or repression of factors involved in tumorantigen signaling or regulation may be used as surrogate measures ofexpression, so long as they are reliably correlated with tumor antigenexpression and/or cancer.

E. Molecular Fingerprint

In some embodiments, the present invention provides “molecularfingerprints” of autoantibodies in cancer. For example, in someembodiments, protein microarrays allow the detection of a plurality ofautoantibodies simultaneously. Such molecular fingerprints provide aprofile of the presence of autoantibodies in particular cancers orcancer sub-types. The profiles find use in providing cancer diagnosesand prognoses. Such prognoses can be used to determine treatment courseof action. For example, in some embodiments, the autoantibody profile ofa particular cancer subtype is indicative of a cancer that is responsiveto a particular choice of therapy. In other embodiments, autoantibodyprofiles are indicative of the aggressiveness of a particular cancersub-type and are used to determine the aggressiveness of treatment to bepursued.

IV. Immunotherapy

The tumor antigens identified during the development of the presentinvention find use in cancer immunotherapy. Such methods areimprovements over the non-specific chemotherapeutic cancer therapiescurrently available. For example, in some embodiments, tumor antigensare used to generate therapeutic antibodies. In other embodiments, thetumor antigens of the present invention find use in the generation ofcancer vaccines.

A. Pharmaceutical Compositions

In some embodiments, the present invention provides pharmaceuticalcompositions that may comprise all or portions of tumor antigenpolynucleotide sequences, tumor antigen polypeptides, inhibitors orantagonists of tumor antigen bioactivity, including antibodies, alone orin combination with at least one other agent, such as a stabilizingcompound, and may be administered in any sterile, biocompatiblepharmaceutical carrier, including, but not limited to, saline, bufferedsaline, dextrose, and water. The pharmaceutical compositions find use astherapeutic agents and vaccines for the treatment of cancer.

The methods of the present invention find use in treating cancers asdescribed in greater detail below. Antibodies can be administered to thepatient intravenously in a pharmaceutically acceptable carrier such asphysiological saline. Standard methods for intracellular delivery ofantibodies can be used (e.g., delivery via liposome). Such methods arewell known to those of ordinary skill in the art. The formulations ofthis invention are usefull for parenteral administration, such asintravenous, subcutaneous, intramuscular, and intraperitoneal.

As is well known in the medical arts, dosages for any one patientdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, sex, time androute of administration, general health, and interaction with otherdrugs being concurrently administered.

Accordingly, in some embodiments of the present invention, compositions(e.g., antibodies and vaccines) can be administered to a patient alone,or in combination with other nucleotide sequences, drugs or hormones orin pharmaceutical compositions where it is mixed with excipient(s) orother pharmaceutically acceptable carriers. In one embodiment of thepresent invention, the pharmaceutically acceptable carrier ispharmaceutically inert. In another embodiment of the present invention,compositions may be administered alone to individuals suffering fromcancer.

Depending on the type of cancer being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration.

For injection, the pharmaceutical compositions of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the presentinvention can be formulated using pharmaceutically acceptable carrierswell known in the art in dosages suitable for oral administration. Suchcarriers enable the pharmaceutical compositions to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For example, aneffective amount of antibody or vaccine may be that amount thatdecreases the presence of cancerous cells (e.g., shrinks or eliminates atumor or reduces the number of circulating cancer cells). Determinationof effective amounts is well within the capability of those skilled inthe art, especially in light of the disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are carbohydrate or protein fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, etc; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, (i.e., dosage).

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients mixed with filler or binderssuch as lactose or starches, lubricants such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. For antibodies to a tumor antigen of the present invention,conditions indicated on the label may include treatment of conditionsrelated to cancer.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Then, preferably, dosage can be formulated in animalmodels (particularly murine models) to achieve a desirable circulatingconcentration range that adjusts antibody levels.

A therapeutically effective dose refers to that amount of antibody thatameliorates symptoms of the disease state. Toxicity and therapeuticefficacy of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds thatexhibit large therapeutic indices are preferred. The data obtained fromthese cell culture assays and additional animal studies can be used informulating a range of dosage for human use. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state; age, weight, and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212,all of which are herein incorporated by reference).

In some embodiments, the pharmaceutical compositions of the presentinvention further include one or more agents useful in the treatment ofcancer. For example, in some embodiments, one or more antibodies orvaccines are combined with a chemotherapeutic agent. Chemotherapeuticagents are well known to those of skill in the art. Examples of suchchemotherapeutics include alkylating agents, antibiotics,antimetabolitic agents, plant-derived agents, and hormones. Among thesuitable alkylating agents are nitrogen mustards, such ascyclophosphamide, aziridines, alkyl alkone sulfonates, nitrosoureas,nonclassic alkylating agents, such as dacarbazine, and platinumcompounds, such as carboplatin and cisplatin. Among the suitableantibiotic agents are dactinomycin, bleomycin, mitomycin C, plicamycin,and the anthracyclines, such as doxorubicin (also known as adriamycin)and mitoxantrone. Among the suitable antimetabolic agents are antifols,such as methotrexate, purine analogues, pyrimidine analogues, such as5-fluorouracil (5-FU) and cytarabine, enzymes, such as theasparaginases, and synthetic agents, such as hydroxyurea. Among thesuitable plant-derived agents are vinca alkaloids, such as vincristineand vinblastine, taxanes, epipodophyllotoxins, such as etoposide, andcamptothecan. Among suitable hormones are steroids. Currently, thepreferred drug is adriamycin. However, other suitable chemotherapeuticagents, including additional agents within the groups of agentsidentified above, may be readily determined by one of skill in the artdepending upon the type of cancer being treated, the condition of thehuman or veterinary patient, and the like.

Suitable dosages for the selected chemotherapeutic agent are known tothose of skill in the art. One of skill in the art can readily adjustthe route of administration, the number of doses received, the timing ofthe doses, and the dosage amount, as needed. Such a dose, which may bereadily adjusted depending upon the particular drug or agent selected,may be administered by any suitable route, including but not limited to,those described above. Doses may be repeated as needed.

B. Antibody Immunotherapy

In some embodiments, the present invention provides therapy for cancercomprising the administration of therapeutic antibodies (See e.g., U.S.Pat. Nos. 6,180,357; and 6,051,230; both of which are hereinincorporated by reference).

In some embodiments, the therapeutic antibodies comprise an antibodygenerated against a tumor antigen of the present invention conjugated toa cytotoxic agent. Such antibodies are particularly suited for targetingtumor antigens expressed on tumor cells but not normal cells. In suchembodiments, a tumor specific therapeutic agent is generated that doesnot target normal cells, thus reducing many of the detrimental sideeffects of traditional chemotherapy. For certain applications, it isenvisioned that the therapeutic agents will be pharmacologic agents willserve as useful agents for attachment to antibodies or growth factors,particularly cytotoxic or otherwise anticellular agents having theability to kill or suppress the growth or cell division of endothelialcells. The present invention contemplates the use of any pharmacologicagent that can be conjugated to an antibody, and delivered in activeform. Exemplary anticellular agents include chemotherapeutic agents,radioisotopes, and cytotoxins. The therapeutic antibodies of the presentinvention may include a variety of cytotoxic moieties, including but notlimited to, radioactive isotopes (e.g., iodine-131, iodine-123,technicium-99m, indium-111, rhenium-188, rhenium-186, gallium-67,copper-67, yttrium-90, iodine-125 or astatine-211), hormones such as asteroid, antimetabolites such as cytosines (e.g., arabinoside,fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycinC), vinca alkaloids (e.g., demecolcine; etoposide; mithramycin), andantitumor alkylating agent such as chlorambucil or melphalan. Otherembodiments may include agents such as a coagulant, a cytokine, growthfactor, bacterial endotoxin or the lipid A moiety of bacterialendotoxin. For example, in some embodiments, therapeutic agents willinclude plant-, fungus- or bacteria-derived toxin, such as an A chaintoxins, a ribosome inactivating protein, α-sarcin, aspergillin,restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin,to mention just a few examples. In some preferred embodiments,deglycosylated ricin A chain is utilized.

In any event, it is proposed that agents such as these may, if desired,be successfully conjugated to an antibody, in a manner that will allowtheir targeting, internalization, release or presentation to bloodcomponents at the site of the targeted tumor cells as required usingknown conjugation technology (See, e.g., Ghose et al., Methods Enzymol.,93:280 [1983]).

For example, in some embodiments the present invention providesimmunotoxins targeted to tumor antigens of the present invention.Immunotoxins are conjugates of a specific targeting agent typically atumor-directed antibody or fragment, with a cytotoxic agent, such as atoxin moiety. The targeting agent directs the toxin to, and therebyselectively kills, cells carrying the targeted antigen. In someembodiments, therapeutic antibodies employ crosslinkers that providehigh in vivo stability (Thorpe et al., Cancer Res., 48:6396 [1988]).

In other embodiments, particularly those involving treatment of solidtumors, antibodies are designed to have a cytotoxic or otherwiseanticellular effect against the tumor vasculature, by suppressing thegrowth or cell division of the vascular endothelial cells. This attackis intended to lead to a tumor-localized vascular collapse, deprivingthe tumor cells, particularly those tumor cells distal of thevasculature, of oxygen and nutrients, ultimately leading to cell deathand tumor necrosis.

In preferred embodiments, antibody based therapeutics are formulated aspharmaceutical compositions and described above. In preferredembodiments, administration of an antibody composition of the presentinvention results in a measurable decrease in cancer (e.g., decrease orelimination of tumor).

C. Cancer Vaccines

In some embodiments, the present invention provides cancer vaccinesdirected against a specific cancer. Cancer vaccines induce a systemictumor-specific immune response. Such a response is capable oferadicating tumor cells anywhere in the body (e.g., metastatic tumorcells). Methods for generating tumor vaccines are well known in the art(See e.g., U.S. Pat. Nos. 5,994,523; 5,972,334; 5,904,920; 5,674,486;and 6,207,147; each of which is herein incorporated by reference).

In some embodiments, tumor vaccines are administered when cancer isfirst detected (e.g., concurrently with other therapeutics such aschemotherapy). In other embodiments, cancer vaccines are administeredfollowing treatment (e.g., surgical resection or chemotherapy) toprevent relapse or metastases. In yet other embodiments, cancer vaccinesare administered prophylactically (e.g., to those at risk of a certaincancer).

In some embodiments, the cancer vaccines of the present inventioncomprise one or more tumor antigens in a pharmaceutical composition(e.g., those described above). In some embodiments, the tumor antigen isinactivated prior to administration. In other embodiments, the vaccinefurther comprises one or more additional therapeutic agents (e.g.,cytokines or cytokine expressing cells).

In some embodiments (e.g., the method described in U.S. Pat. No.5,674,486, herein incorporated by reference), selected cells from apatient, such as fibroblasts, obtained, for example, from a routine skinbiopsy, are genetically modified to express one or more cytokines.Alternatively, patient cells that may normally serve as antigenpresenting cells in the immune system such as macrophages, monocytes,and lymphocytes may also be genetically modified to express one or morecytokines. The cytokine expressing cells are then mixed with thepatient's tumor antigens (e.g., a tumor antigen of the presentinvention), for example in the form of irradiated tumor cells, oralternatively in the form of purified natural or recombinant tumorantigen, and employed in immunizations, for example subcutaneously, toinduce systemic anti-tumor immunity.

The vaccines of the present invention may be administered using anysuitable method, including but not limited to, those described above. Inpreferred embodiments, administration of a cancer vaccine of the presentinvention results in elimination (e.g., decrease or elimination oftumors) or prevention of detectable cancer cells.

V. Other Therapies

The present invention is not limited to the therapeutic applicationsdescribed above. Indeed, any therapeutic application that specificallytargets tumor cells expressing the tumor antigens of the presentinvention are contemplated, including but not limited to, antisensetherapies.

For example, in some embodiments, the present invention employscompositions comprising oligomeric antisense compounds, particularlyoligonucleotides, for use in modulating the function of nucleic acidmolecules encoding tumor antigens of the present invention, ultimatelymodulating the amount of tumor antigen produced. This is accomplished byproviding antisense compounds that specifically hybridize with one ormore nucleic acids encoding tumor antigens. The specific hybridizationof an oligomeric compound with its target nucleic acid interferes withthe normal function of the nucleic acid. This modulation of function ofa target nucleic acid by compounds that specifically hybridize to it isgenerally referred to as “antisense.” The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity that may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of tumorantigens. In the context of the present invention, “modulation” meanseither an increase (stimulation) or a decrease (inhibition) in theexpression of a gene. For example, expression may be inhibited topotentially prevent tumor proliferation or stimulated to increase acancer-specific immune response (e.g., as a cancer vaccine).

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of the present invention, is a multistep process. The processusually begins with the identification of a nucleic acid sequence whosefunction is to be modulated. This may be, for example, a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. In the present invention, the target is a nucleicacid molecule encoding a tumor antigen of the present invention. Thetargeting process also includes determination of a site or sites withinthis gene for the antisense interaction to occur such that the desiredeffect, e.g., detection or modulation of expression of the protein, willresult. Within the context of the present invention, a preferredintragenic site is the region encompassing the translation initiation ortermination codon of the open reading frame (ORF) of the gene. Since thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). Eukaryotic and prokaryotic genes may have two or morealternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of the presentinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAmolecule transcribed from a gene encoding a tumor antigen of the presentinvention, regardless of the sequence(s) of such codons.

Translation termination codon (or “stop codon”) of a gene may have oneof three sequences (i.e., 5′-UAA, 5′-UAG and 5′-UGA; the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region” and “translation initiation codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. Similarly, the terms “stop codonregion” and “translation termination codon region” refer to a portion ofsuch an mRNA or gene that encompasses from about 25 to about 50contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon.

The open reading frame (ORF) or “coding region,” which refers to theregion between the translation initiation codon and the translationtermination codon, is also a region that may be targeted effectively.Other target regions include the 5′ untranslated region (5′ UTR),referring to the portion of an mRNA in the 5′ direction from thetranslation initiation codon, and thus including nucleotides between the5′ cap site and the translation initiation codon of an mRNA orcorresponding nucleotides on the gene, and the 3′ untranslated region(3′ UTR), referring to the portion of an mRNA in the 3′ direction fromthe translation termination codon, and thus including nucleotidesbetween the translation termination codon and 3′ end of an mRNA orcorresponding nucleotides on the gene. The 5′ cap of an mRNA comprisesan N7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” that are excised from atranscript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites (i.e., intron-exonjunctions) may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen that are sufficiently complementary to the target (i.e.,hybridize sufficiently well and with sufficient specificity) to give thedesired effect. For example, in preferred embodiments of the presentinvention, antisense oligonucleotides are targeted to or near the startcodon.

In the context of this invention, “hybridization,” with respect toantisense compositions and methods, means hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases. For example, adenine andthymine are complementary nucleobases that pair through the formation ofhydrogen bonds. It is understood that the sequence of an antisensecompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. An antisense compound isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired (i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed).

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with specificity, can be used to elucidate thefunction of particular genes. Antisense compounds are also used, forexample, to distinguish between functions of various members of abiological pathway.

The specificity and sensitivity of antisense is also applied fortherapeutic uses. For example, antisense oligonucleotides have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man. Antisense oligonucleotides have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides areuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues, and animals,especially humans.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases(i.e., from about 8 to about 30 linked bases), although both longer andshorter sequences may find use with the present invention. Particularlypreferred antisense compounds are antisense oligonucleotides, even morepreferably those comprising from about 12 to about 25 nucleobases.

Specific examples of preferred antisense compounds useful with thepresent invention include oligonucleotides containing modified backbonesor non-natural internucleoside linkages. As defined in thisspecification, oligonucleotides having modified backbones include thosethat retain a phosphorus atom in the backbone and those that do not havea phosphorus atom in the backbone. For the purposes of thisspecification, modified oligonucleotides that do not have a phosphorusatom in their internucleoside backbone can also be considered to beoligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e., the backbone) of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science 254:1497 (1991).

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂, —NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta 78:486[1995]) i.e., an alkoxyalkoxy group. A further preferred modificationincludes 2′-dimethylaminooxyethoxy (i.e., a O(CH₂)₂ON(CH₃)₂ group), alsoknown as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808. Certainof these nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2. degree ° C. andare presently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the present inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates that enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, (e.g., hexyl-S-tritylthiol), a thiocholesterol, analiphatic chain, (e.g., dodecandiol or undecyl residues), aphospholipid, (e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or apolyethylene glycol chain or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

One skilled in the relevant art knows well how to generateoligonucleotides containing the above-described modifications. Thepresent invention is not limited to the antisensce oligonucleotidesdescribed above. Any suitable modification or substitution may beutilized.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds that are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of the presentinvention, are antisense compounds, particularly oligonucleotides, whichcontain two or more chemically distinct regions, each made up of atleast one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNaseH is a cellular endonuclease thatcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotides when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the present invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above.

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption to generatepharmaceutical compositions as described above.

VI. Transgenic Animals Expressing Exogenous Genes and Variants Thereof

The present invention contemplates the generation of transgenic animalscomprising an exogenous tumor antigen gene of the present invention ormutants and variants thereof (e.g., truncations). In preferredembodiments, the transgenic animal displays an altered phenotype (e.g.,increased presence of tumor antigens) as compared to wild-type animals.Methods for analyzing the presence or absence of such phenotypes includebut are not limited to, those disclosed herein. In some preferredembodiments, the transgenic animals further display an increased growthof tumors or increased evidence of cancer.

The transgenic animals of the present invention find use in drug (e.g.,cancer therapy) screens. In some embodiments, test compounds (e.g., adrug that is suspected of being useful to treat cancer) and controlcompounds (e.g., a placebo) are administered to the transgenic animalsand the control animals and the effects evaluated. In other embodiments,transgenic and control animals are given immunotherapy (e.g., includingbut not limited to, the methods described above) and the effect oncancer symptoms is assessed.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter that allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Stewart, et al.,EMBO J., 6:383 [1987]). Alternatively, infection can be performed at alater stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner et al., Nature 298:623 [1982]). Most of the founderswill be mosaic for the transgene since incorporation occurs only in asubset of cells that form the transgenic animal. Further, the foundermay contain various retroviral insertions of the transgene at differentpositions in the genome that generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into thegermline, albeit with low efficiency, by intrauterine retroviralinfection of the midgestation embryo (Jahner et al., supra [1982]).Additional means of using retroviruses or retroviral vectors to createtransgenic animals known to the art involve the micro-injection ofretroviral particles or mitomycin C-treated cells producing retrovirusinto the perivitelline space of fertilized eggs or early embryos (PCTInternational Application WO 90/08832 [1990], and Haskell and Bowen,Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley etal., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065[1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch, Science 240:1468 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

In still other embodiments, homologous recombination is utilized toknock-out gene function or create deletion mutants (e.g., truncationmutants). Methods for homologous recombination are described in U.S.Pat. No. 5,614,396, incorporated herein by reference.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); ° C. (degrees Centigrade); and Sigma (Sigma Chemical Co.,St. Louis, Mo.).

EXAMPLE 1

Detection of Autoantibodies to UCH-L3

This Example describes the detection of autoantibodies to UCH-L3 in theserum of patients with colon cancer.

A. Materials and Methods

Serum Samples

Following informed consent, sera were obtained at the time of diagnosisfrom 45 patients with colon cancer, 24 patients with lung cancer, 20with inflammatory bowel disease, 15 with colon adenoma and from 15healthy individuals.

Cell Lines and Cell Culture

The LoVo colon adenocarcinoma cell line was cultured (6% CO₂, 37° C.) inDMEM medium containing 10% fetal bovine serum, penicillin (100units/ml), and streptomycin (100 units/ml), all purchased fromInvitrogen. The cells were solubilized in lysis buffer (Wall et al.,Analytical Chemistry 72, 1099-1111 [2000]) containing 6 M urea, 2 Mthiourea, 1.0% n-octyl β-D-glucopyranoside, 2 mM dithioerythritol,protease inhibitor cocktail (Boehringer, Manheim, Germany), and 2%ampholytes, pH 3.5-10 (Bio-Rad). The lysates were scraped, RNase A (10U/ml) and DNase I (100 U/ml) were added, and the solution left on icefor 30 min. The supernatant was vortexed, clarified by centrifugation(20,000×g for 10 min), and collected.

Protein Fractionation

A preparative-scale Rotofor (Bio-Rad) was used to separate proteins inthe first dimension (Madoz-Gurpide et al., Proteomics 1, 1279-1287[2001]). Whole cell protein extracts were diluted to 55 ml with runningbuffer (the same buffer as the lysis buffer except that 0.5% n-octylβ-D-glucopyranoside was used), and separated by isoelectric focusing for6 hr (10° C.). 20 separate fractions were collected. The proteinconcentration and pH of each fraction was measured as previouslydescribed (Madoz-Gurpide et al., 2001, supra).

The high-resolution liquid chromatography (HPLC) reversed phase columnR2/H (Applied Biosystems) was used for the separation of proteins in thesecond dimension. Separations were performed at a flow rate of 1.3ml/min using water/acetonitrile gradients (solvent A: 98% H₂O, 2%acetonitrile, 0.1% TFA; solvent B: 90% acetonitrile, 10% H₂O, 0.1% TFA).The gradient profile used was as follows: (0) 95% solvent A for 2.5 min;(1) 95 to 75% A in 2.5 min; (2) 75 to 35% A in 40 min; (3) 35% A for 5min; (4) 35 to 15% A in 5 min; (5) 15 to 5% A in 5 min; (6) 5 to 95% in5 min. Protein fractions were collected every 30 seconds (88 fractionsfrom each 1D fraction) starting 10 min into the gradient, thenimmediately frozen at −80° C. The fractions were lyophilized undervacuum, and resuspended in 25 μl of 60% PBS, 40% glycerol.

Protein Microarrays

3968 features were prepared that consisted of 1760 distinct LoVofractions in duplicate, 64 positive, and 384 negative controls, andarrayed onto nitrocellulose membranes supported on glass slides(Schleicher and Schuell) using a 32-pin Flexys arrayer, as previouslydescribed (Madoz-Gurpide et al., 2001, supra). Biotinylated BSA wasprinted to act as a “landmark” to orient the arrays.

Patient serum was analyzed with the microarrays. Each slide was placedin its hybridization chamber inside a GeneTAC Hybridization Station(Genomic Solutions). 100 μL serum was added at a 1:50 dilution inblocking solution (PBS containing 3% non-fat dry milk) as a source ofprimary antibody, and allowed to hybridize for 2 h at 22° C. underagitation. The microarrays were washed four times in PBST (PBS, 0.1%Tween-20) for 1 min, followed by another two 1 min-cycles of washing inPBS. Biotinylated anti-human IgG (Amersham) was introduced into thehybridization chamber at a dilution of 1:20 in blocking solution.Following a 1 hr incubation, the membranes were washed in PBST fourtimes for 1 min, and twice in PBS for 1 min. Streptavidin,R-phycoerythrin (Molecular Probes, Eugene, Oreg.) was added at adilution 1:100 for 20 min. The slides were washed four times in PBST for1 min, two times in PBS for 1 min, and then centrifuged at 200×g todryness. The microarrays were imaged at 550 nm using a GeneTAC LS-IVscanner (Genomic Solutions).

Analysis of Protein Microarray Images

Scanned microarrays were analyzed using the GeneTAC Biochip Analyzersoftware package (Genomic Solutions). Images were manually fitted with agrid and the spot intensities were measured. The local background wassubtracted from the signal at each spot, and the resulting averageintensity of each spot pixels determined. Spots and/or areas withobvious defects such as signal lower than background or high backgroundwere excluded from subsequent analysis.

Mathematical and Statistical Analysis of Antibody Reactivity

Variable signal brightness between slides was adjusted by subtractingfrom each average intensity value the 25^(th) percentile of theintensity measures in each patch (a rectangular area of dots printed bythe same pin). Resulting intensity measures less than 10 were set to 10.Differences between batches of slides that were printed and hybridizedas groups were observable in the data (4 batches), so the patch-adjustedintensity values were compared to the median of the values for normalsamples, and dots were categorized as positively reacting if thisrelative reactivity was at least 2.0. Using this categorization,one-sided Chi-square tests comparing colon vs. normal and lung vs.normal were performed.

Protein Identification by Mass Spectrometry

2-D-RPLC fractions were solubilized (1:1 PBS and NH₄HCO₃), thensubjected to trypsin digestion at 37° C. for 18 h. Proteinidentifications were performed by nano-flow capillary liquidchromatography coupled with electrospray quadrupole-time of flighttandem mass spectrometry (LC ESI Q-TOF MS/MS) using a Q-TOF Micro(Micromass, Manchester, UK). ESI MS/MS tandem spectra were recorded inthe automated MS to MS/MS switching mode, with m/z-dependent set ofcollision offset values. Doubly and triply charged ions were selectedand fragmented using argon as the collision gas. The acquired spectrawere processed and searched against the non-redundant Swiss-Prot proteinsequence database using ProteinLynx Global Server (available fromMicromass).

High-Density Oligonucleotide Microarrays

High-density oligonucleotide microarrays (Affymetrix, Santa Clara,Calif.) were probed as previously described (Giordano et al., AmericanJournal of Pathology 159, 1231-1238 [2001]). The arrays were scannedusing the GeneArray scanner (Affymetrix). Image analysis was performedwith GeneChip 4.0 software (Affymetrix). Expression values werecalculated as previously described (Schwartz et al., Cancer Research 62,4722-4729 [2002]).

2-D Polyacrylamide Gel Electrophoresis and Western Blotting

The procedure followed was as described previously (Strahler et al.,1989 Two-dimensional electrophoresis. In: Protein Structure. A PracticalApproach., T. Creighton, ed. (England: IRL Press)). Proteins were run inthe first dimension by IEF. For the second dimension separation, agradient of 11-14% acrylamide (Crescent Chemical, Hauppauge, N.Y.) wasused. Proteins were transferred to an Immobilon-P PVDF membrane(Millipore, Bedford, Mass.) or visualized by silver staining of thegels. The membranes were incubated with sera at a 1:200 dilution, andwere then incubated with HRP-conjugated IgG antibodies (Amersham) at adilution of 1:1000. Immunodetection was accomplished by ECL (Amersham).Patterns visualized were compared directly with Coomassie blue-stainedblots from the same sample to determine correlation with proteinpatterns. An anti-UCH-L3 antibody (obtained from Dr. Keith Wilkinson,Emory University, Ga.) was used at a 1:10,000 dilution on Western blotsin order to detect UCH-L3.

B. Results

Protein Microarray Based Assay for Autoantibodies in Sera from Patientswith Colon Cancer

Preparative quantities (approximately 500 mg) of solubilized proteinsisolated from the LoVo colon adenocarcinoma cell line were resolved byRotofor isoelectric focusing in the first dimension (Madoz-Gurpide etal., 2001, supra). Following a 6-hr isoelectric focusing separationperiod, 20 fractions covering the pl range of 3.5-10 were collected inpolypropylene tubes by vacuum harvesting. Each Rotofor fraction wasseparated in the second dimension by reverse-phase liquid chromatographyinto 88 fractions, for a total of 1760 fractions. All fractions werelyophilized to dryness, resuspended in 25 μl PBS/glycerol, and then usedto prepare protein microarrays. The volume of sample that every pin inthe robot head can deliver is approximately 0.5 nL. Considering that theaverage concentration of total protein in each fraction wasapproximately 2.3 μg/μl and given that each reverse-phase fractioncontained 1-10 proteins as identified by mass spectrometry, anestimation of the amount of individual proteins in each dot isapproximately 200 pg with a wide range.

3698 features were arrayed on each slide, representing 1760 separateprotein fractions in duplicate, from the LoVo colon adenocarcinoma cellline, as well as positive, negative, and landmark controls. Fifteen serafrom colon cancer patients, 15 sera from lung cancer patients and 15control sera from healthy subjects were individually hybridized to theLoVo protein microarrays. Scanned images were quantitatively analyzedfor intensity of hybridization of spotted individual fractions with eachserum. Control spots on the microarrays, including tetanus toxoid, humanIgG, and biotinylated protein controls repeatedly showed similarreactivity in all sera assayed. A set of 39 of 1760 fractions showedgreater reactivity with sera from colon cancer patients relative tohealthy controls (p<0.01). Twenty four of 1760 fractions showed greaterreactivity with sera from lung cancer patients relative to healthycontrols (p<0.01). Only 5 fractions showed greater reactivity with serafrom both colon and lung patients relative to healthy controls.

Identification of UCH-L3 Protein in Fraction L04428 by Mass Spectrometry

Among the 39 fractions that demonstrated greater reactivity with coloncancer sera, the most reactive fraction (L04428) exhibited reactivitywith 9 out of 15 colon cancer sera. Given the distinctive pattern ofreactivity of fraction L04428, a tryptic digest of the proteinconstituents of this fraction was prepared and subjected toidentification by tandem mass spectrometry (ESI-Q-TOF). Analysis offraction L04428 revealed that it contained the ubiquitincarboxyl-terminal hydrolase isozyme 3 (UCH-L3) protein. The precursorion m/z 949.9609 resulting from the tryptic digest matched with a16-aminoacid sequence of UCH-L3 against protein sequence database withgood accuracy (error=0.09 Daltons).

High Level Expression of the UCH-L3 Gene and Protein in ColonAdenocarcinoma

To confirm the presence of UCH-L3 in fraction L04428, LoVo proteinmicroarrays were probed with a rabbit anti-UCH-L3 antibody. Specificreactivity for fraction L04428 was found. The expression of UCH-L1 andL3 was further explored in different tumor types. UCH-L3 and L1 geneexpression was analyzed in 329 tissue samples, consisting of 51 colonadenocarcinomas, 91 lung adenocarcinomas, 10 pancreatic tumors, 73 braintumors and 104 ovarian tumors using DNA microarrays. UCH-L3 was found tobe expressed at 3-5 fold higher levels in colon tumors than thatobserved in all other tumor types examined (p<0.01). In contrast, theUCH-L1 gene, whose protein product was found to be the target ofautoantibodies in lung cancer (Brichory et al., 2001, supra), was highlyexpressed in lung cancer relative to colon cancer (p<0.001). Expressionof these two genes was further examined in the LoVo colon adenocarcinomaand the A549 lung adenocarcinoma cell lines. The LoVo cell lineexpressed high levels of the UCH-L3 gene product, but did not expressUCH-L1, whereas the A549 cell line expressed both the UCH-L1 and UCH-L3gene products. The results obtained for UCH-L1 and UCH-L3 expression intumors and cell lines using DNA microarrays were confirmed by real timePCR.

Validation of the Presence and Specificity of UCH-L3 Autoantibodies inSera from Colon Cancer Patients by Western Blot Analysis

The occurrence of autoantibodies to UCH-L3 in colon cancer and thespecificity of the antibody response to UCH-L3 was examined by Westernblot analysis of sera from patients with colon adenocarcinomas,inflammatory bowel disease, colon adenoma or lung cancer and of serafrom healthy subjects. To this end, solubilized proteins from the LoVocell line were resolved by 2-D PAGE and transferred onto Immobilon-PPVDF membranes. In order to identify the location of UCH-L3 by 2-D PAGE,the LoVo cell blots were hybridized with a rabbit anti-UCH-L3 antibody.A highly reactive protein spot was found with an estimated molecularweight of 26 kDa and with a pI of 4.7, concordant with the predictedmass and pI of UCH-L3. The protein was excised from silver-stained gels,subjected to tryptic digestion and identified as UCH-L3 by Q-TOF tandemmass spectrometry. 2-D Western blots of LoVo cell proteins were preparedand hybridized with different subject sera. Sera from 19/43 patientswith colon cancer exhibited IgG-based reactivity against UCH-L3. Incontrast, none of the sera from 15 healthy, 15 colon adenoma and 24 lungcancer sera were reactive with UCH-L3 (Table 1). Only 2/20 sera obtainedfrom patients with inflammatory bowel disease exhibited immunoreactivityagainst UCH-L3. 13 colon cancer sera were analyzed by both proteinmicroarray and by 2D Western blots. Ten of the 13 sera showed concordantresults between the two methods. Taken together, autoantibodies toUCH-L3 protein exhibited a high degree of specificity to colon cancer.

TABLE 1 Anti-UCH-L3 IgG in patient sera. UCH-L3 autoantibody Serumnumber of subjects positive Normal 15 0 Adenoma patient 15 0 IBD patient20 2 Colon cancer patient* 43 19 Lung cancer patient 24 0 *Colon cancersamples were positive more frequently than the others (P = 0.0002 for aFisher's exact test).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

1. A method for identifying subjects suspected of having colorectalcancer, comprising: a) providing a serum sample from a subject; and b)detecting the presence or absence of an autoantibody to ubiquitincarboxyl-terminal hydrolase isozyme 3 (UCH-L3) in said serum sample,wherein subjects having said a serum antibody to UCH-L3 are suspected ofhaving colorectal cancer.
 2. The method of claim 1, wherein said subjectis a human subject.